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Quantitative X-ray phase contrast waveguide imaging of bacterial endospores.

Wilke RN, Hoppert M, Krenkel M, Bartels M, Salditt T - J Appl Crystallogr (2015)

Bottom Line: The quantitative phase maps obtained by iterative phase retrieval using a modified hybrid input-output algorithm allow for mass and mass density determinations on the level of single individual endospores but include also large field of view investigations.Depending on the field of view and method, a resolution down to 65 nm was achieved at a maximum applied dose of below 5 × 10(5) Gy.Masses in the range of about ∼110-190 (20) fg for isolated endospores have been obtained.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Göttingen, Institute for X-ray Physics, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany.

ABSTRACT

Quantitative waveguide-based X-ray phase contrast imaging has been carried out on the level of single, unstained, unsliced and freeze-dried bacterial cells of Bacillus thuringiensis and Bacillus subtilis using hard X-rays of 7.9 keV photon energy. The cells have been prepared in the metabolically dormant state of an endospore. The quantitative phase maps obtained by iterative phase retrieval using a modified hybrid input-output algorithm allow for mass and mass density determinations on the level of single individual endospores but include also large field of view investigations. Additionally, a direct reconstruction based on the contrast transfer function is investigated, and the two approaches are compared. Depending on the field of view and method, a resolution down to 65 nm was achieved at a maximum applied dose of below 5 × 10(5) Gy. Masses in the range of about ∼110-190 (20) fg for isolated endospores have been obtained.

No MeSH data available.


(a) The CTF-based reconstruction of region (A) of the B. subtilis sample. Three isolated endospores can be seen. (b), (c) Calculated gradients (filtered with  pixel Gaussian) in the horizontal and vertical directions of the region of two endospores of (a). The black arrow heads highlight the transition between two different structural regions of the endospore that are attributed to the coat and the inner part. The repetitive structure on the gradient maps is an artefact of the reconstruction in (a). The scale bars denotes 0.5 µm.
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fig10: (a) The CTF-based reconstruction of region (A) of the B. subtilis sample. Three isolated endospores can be seen. (b), (c) Calculated gradients (filtered with  pixel Gaussian) in the horizontal and vertical directions of the region of two endospores of (a). The black arrow heads highlight the transition between two different structural regions of the endospore that are attributed to the coat and the inner part. The repetitive structure on the gradient maps is an artefact of the reconstruction in (a). The scale bars denotes 0.5 µm.

Mentions: Finally, the phase reconstructions using the CTF-based method follow. Figs. 9 ▶(a) and 9 ▶(b) show reconstructions of the B. thuringiensis sample at final magnifications and using four and two different planes , respectively. The visual impression of the images is quite good in comparison to the mHIO reconstructions of Figs. 7 ▶(a) and 7 ▶(b). In particular, the noise in the data is significantly reduced, which makes it easier for the eye to identify the cells and intracellular components such as endospores. However, the overall phase shift deviates slightly from the preceding iterative mHIO reconstructions due to a possibly non-optimum choice of regularization parameters. An inspection of the PSDs suggests an overall achieved resolution of about 135 nm for the data of Fig. 9 ▶(a) and of about 100 nm for the data of Fig. 9 ▶(b) (cf. Fig. 6 ▶c). In comparison to the mHIO reconstructions, the slightly improved resolution is in agreement with the applied doses of about  Gy, which are by about one order of magnitude higher for the case of the reconstruction scheme based on the CTF (cf. Table 1 ▶). The reconstruction of Fig. 9 ▶(b) may still be improved as here only data sets from two planes (instead of four) were available. The reconstruction of region (A) of the B. subtilis sample of dried endospores is presented in Fig. 10 ▶(a) (final magnification ). The image shows the three endospores of the mHIO reconstruction in Fig. 8 ▶(a) with a clearly reduced noise level. Here, the PSD of the region with the two endospores (2) and (3) (Fig. 6 ▶d) indicates a resolution down to about 65 nm (half-period), which is in good agreement with the improved visual impression. The contrast of the phase map clearly allows us to identify two regions within the spore (2) that may be linked to the coat and the interior, namely the cortex and the core. These two structural regions of the endospore can be better distinguished in Figs. 10 ▶(b) and 10 ▶(c), which show the horizontal and vertical gradients (filtered with  pixel Gaussian). Size measurements from the reconstruction of Fig. 10 ▶(a) yield about 270 nm for the interior and 170–200 nm for the outer part (coat) along the horizontal direction.


Quantitative X-ray phase contrast waveguide imaging of bacterial endospores.

Wilke RN, Hoppert M, Krenkel M, Bartels M, Salditt T - J Appl Crystallogr (2015)

(a) The CTF-based reconstruction of region (A) of the B. subtilis sample. Three isolated endospores can be seen. (b), (c) Calculated gradients (filtered with  pixel Gaussian) in the horizontal and vertical directions of the region of two endospores of (a). The black arrow heads highlight the transition between two different structural regions of the endospore that are attributed to the coat and the inner part. The repetitive structure on the gradient maps is an artefact of the reconstruction in (a). The scale bars denotes 0.5 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4379437&req=5

fig10: (a) The CTF-based reconstruction of region (A) of the B. subtilis sample. Three isolated endospores can be seen. (b), (c) Calculated gradients (filtered with  pixel Gaussian) in the horizontal and vertical directions of the region of two endospores of (a). The black arrow heads highlight the transition between two different structural regions of the endospore that are attributed to the coat and the inner part. The repetitive structure on the gradient maps is an artefact of the reconstruction in (a). The scale bars denotes 0.5 µm.
Mentions: Finally, the phase reconstructions using the CTF-based method follow. Figs. 9 ▶(a) and 9 ▶(b) show reconstructions of the B. thuringiensis sample at final magnifications and using four and two different planes , respectively. The visual impression of the images is quite good in comparison to the mHIO reconstructions of Figs. 7 ▶(a) and 7 ▶(b). In particular, the noise in the data is significantly reduced, which makes it easier for the eye to identify the cells and intracellular components such as endospores. However, the overall phase shift deviates slightly from the preceding iterative mHIO reconstructions due to a possibly non-optimum choice of regularization parameters. An inspection of the PSDs suggests an overall achieved resolution of about 135 nm for the data of Fig. 9 ▶(a) and of about 100 nm for the data of Fig. 9 ▶(b) (cf. Fig. 6 ▶c). In comparison to the mHIO reconstructions, the slightly improved resolution is in agreement with the applied doses of about  Gy, which are by about one order of magnitude higher for the case of the reconstruction scheme based on the CTF (cf. Table 1 ▶). The reconstruction of Fig. 9 ▶(b) may still be improved as here only data sets from two planes (instead of four) were available. The reconstruction of region (A) of the B. subtilis sample of dried endospores is presented in Fig. 10 ▶(a) (final magnification ). The image shows the three endospores of the mHIO reconstruction in Fig. 8 ▶(a) with a clearly reduced noise level. Here, the PSD of the region with the two endospores (2) and (3) (Fig. 6 ▶d) indicates a resolution down to about 65 nm (half-period), which is in good agreement with the improved visual impression. The contrast of the phase map clearly allows us to identify two regions within the spore (2) that may be linked to the coat and the interior, namely the cortex and the core. These two structural regions of the endospore can be better distinguished in Figs. 10 ▶(b) and 10 ▶(c), which show the horizontal and vertical gradients (filtered with  pixel Gaussian). Size measurements from the reconstruction of Fig. 10 ▶(a) yield about 270 nm for the interior and 170–200 nm for the outer part (coat) along the horizontal direction.

Bottom Line: The quantitative phase maps obtained by iterative phase retrieval using a modified hybrid input-output algorithm allow for mass and mass density determinations on the level of single individual endospores but include also large field of view investigations.Depending on the field of view and method, a resolution down to 65 nm was achieved at a maximum applied dose of below 5 × 10(5) Gy.Masses in the range of about ∼110-190 (20) fg for isolated endospores have been obtained.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Göttingen, Institute for X-ray Physics, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany.

ABSTRACT

Quantitative waveguide-based X-ray phase contrast imaging has been carried out on the level of single, unstained, unsliced and freeze-dried bacterial cells of Bacillus thuringiensis and Bacillus subtilis using hard X-rays of 7.9 keV photon energy. The cells have been prepared in the metabolically dormant state of an endospore. The quantitative phase maps obtained by iterative phase retrieval using a modified hybrid input-output algorithm allow for mass and mass density determinations on the level of single individual endospores but include also large field of view investigations. Additionally, a direct reconstruction based on the contrast transfer function is investigated, and the two approaches are compared. Depending on the field of view and method, a resolution down to 65 nm was achieved at a maximum applied dose of below 5 × 10(5) Gy. Masses in the range of about ∼110-190 (20) fg for isolated endospores have been obtained.

No MeSH data available.